Schisandrin B induced antioxidant response is partly mediated by cytochrome P-4502E1 catalyzed reaction in mouse liver

In order to explore the role of cytochrome P-450 (CYP) 2E1 in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effects of Sch B treatment on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression were compared between wild-type and cyp2e1 knock-out C57B/6N mice. Cyp2e1 knock-out mice exhibited a significantly smaller degree of Sch B-induced enhancement in hepatic mtGAS when compared with the wild-type counterpart. But Hsp25/70 expression induced by Sch B was not affected. Sch B-induced enhancement of mtGAS was corroborated by the increase in hepatic mitochondrial antioxidant capacity, as assessed by in vitro measurement of oxidant production, with the enhancing effect being slightly reduced in the knock-out mice. Using liver microsomes prepared from wild-type and knock-out mice as a source of CYP, Sch B was found to be a good co-substrate for the CYP-catalyzed reaction, with the rate of NADPH oxidation observable in microsomes prepared from knock-out mice being slower. The CYP-catalyzed reaction with Sch B was associated with a concomitant production of oxidant species, with the extent of oxidant production being reduced in cyp2e1 knock-out mouse microsomes. Taken together, the results indicate that CYP2E1 is partly responsible for the hepatic metabolism of Sch B that may trigger the antioxidant response in vivo.     

Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.     

Coplanar polychlorinated biphenyl-induced CYP1A1 is regulated through caveolae signaling in vascular endothelial cells

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that can induce inflammatory processes in the vascular endothelium. We hypothesize that the plasma membrane microdomains called caveolae are critical in endothelial activation and toxicity induced by PCBs. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. We focused on the role of caveolae and their major protein component, caveolin-1 (Cav-1), on aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 1A1 (CYP1A1) by coplanar PCBs. Endothelial cell exposure to PCB77 increased both caveolin-1 and CYP1A1 levels in a time-dependent manner in total cell lysates, with a maximum increase at 6h. Furthermore, PCB77 accumulated mainly in the caveolae-rich fraction, as determined by gas chromatograph-mass spectrometry. Immunoprecipitation analysis revealed that PCB77 increased AhR binding to caveolin-1. Silencing of caveolin-1 significantly attenuated PCB77-mediated induction of CYP1A1 and oxidative stress. Similar effects were observed in caveolin-1 null mice treated with PCB77. These data suggest that caveolae may play a role in regulating vascular toxicity induced by persistent environmental pollutants such as coplanar PCBs. This may have implications in understanding mechanisms of inflammatory diseases induced by environmental pollutants.     

Regulatory factors involved in species-specific modulation of arylhydrocarbon receptor (AhR)-dependent gene expression in humans and mice

The arylhydrocarbon receptor (AhR) mediates toxicities of dioxins, including the most potent congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), by being translocated to the nucleus upon ligand-binding and inducing expression of target genes. Although the species-specific activity of the AhR is primarily attributable to species-specific AhR-ligand affinity, the precise mechanism has not been clarified. We investigated the modulation mechanisms of AhR in Hepa1c1c7 and HepG2 hepatoma cells, which were derived from high-affinity-AhR-expressing C57BL/6 mice and low-affinity-AhR-expressing humans, respectively. Although, consistent with their AhR affinities, TCDD induced a greater amount of cytochrome P450 1A1 (CYP1A1) mRNA, one of the most sensitive AhR-targets, in Hepa1c1c7 cells than in HepG2 cells immediately after exposure, both cells expressed a similar level of CYP1A1 mRNA from 4 h onward. A rapid decrease in the AhR protein after nuclear translocation in Hepa1c1c7 cells was suggested to contribute to suppression of CYP1A1 induction to the same level as in HepG2 cells. Different profiles of histone deacetylase 1 (HDAC1)-binding to the CYP1A1 promoter and histone acetylation between both cell lines and lower degradation rate of CYP1A1 mRNA in HepG2 cells were also implicated in regulating their target gene expression. These factors have been highly suggested to be involved in the species-specific modulation mechanism of AhR function.     

Functional Expression of House Fly (Musca domestica) Cytochrome P450 CYP6D1 in Yeast (Saccharomyces cerevisiae)

Cytochrome P450 CYP6D1 from the house fly is important in the detoxication of xenobiotics and in resistance to pyrethroid insecticides. In house fly microsomes CYP6D1 requires cytochrome b₅ for the metabolism of some substrates, such as benzo[a]pyrene, but does not require cytochrome b₅ for the metabolism of other substrates such as methoxyresorufin. To examine the molecular mechanisms involved in its metabolism of pyrethroids and other substrates, a system for the heterologous expression of CYP6D1 in the yeast Saccharomyces cerevisiae was developed. Heterologous CYP6D1 can be inducibly expressed by culture in media with galactose as the sole carbon source, and is successfully inserted into the yeast microsomes. CYP6D1 is enzymatically active, as measured by methoxyresorufin-O-demethylation, indicating that CYP6D1 is able to interact with yeast P450 reductase. However, CYP6D1 expression did not result in measurable benzo[a]pyrene hydroxylation, suggesting that CYP6D1 cannot interact with yeast cytochrome b₅, or that there is insufficient cytochrome b₅ in the yeast microsomes to support this CYP6D1-mediated activity. Some suggestions are made for improving the yeast microsomal oxidoreductase environment in order to optimize CYP6D1 function.     

Coexpression of genetically engineered fused enzyme between yeast NADPH-P450 reductase and human cytochrome P450 3A4 and human cytochrome b5 in yeast

Human hepatic cytochrome P450 3A4 (CYP3A4) was expressed in yeast Saccharomyces cerevisiae. While the expression level was high as compared with other human hepatic cytochrome P450s, CYP3A4 showed almost no catalytic activity toward testosterone. Coexpression of CYP3A4 with yeast NADPH-P450 reductase did not give a full activity. Low monooxygenase activity of CYP3A4 was attributed to the insufficient reduction of heme iron of CYP3A4 by NADPH-P450 reductase. To enhance the efficiency of electron transfer from NADPH-P450 reductase to CYP3A4, a fused enzyme was constructed between CYP3A4 and yeast NADPH-P450 reductase. The rapid reduction of the heme iron of the fused enzyme by NADPH was observed. The fused enzyme showed a high testosterone 6beta-hydroxylation activity with a sigmoidal velocity saturation curve. However, the coupling efficiency between NADPH utilization and testosterone 6beta-hydroxylation was only 10%. Finally, coexpression of the fused enzyme and human cytochrome b5 was examined. A significant decrease in the Km value and a remarkable increase in the coupling efficiency were observed. Substrate-induced spectra revealed that the dissociation constant of the fused enzyme for testosterone significantly decreased with coexpression of human cytochrome b5. These results strongly suggest that human cytochrome b5 directly interacts with the CYP3A4 domain of the fused enzyme and modifies the tertiary structure of substrate binding pocket, resulting in tight binding of the substrate and high coupling efficiency.     

Common Commercial and Consumer Products Contain Activators of the Aryl Hydrocarbon (Dioxin) Receptor

Activation of the Ah receptor (AhR) by halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), can produce a wide variety of toxic and biological effects. While recent studies have shown that the AhR can bind and be activated by structurally diverse chemicals, how widespread of these AhR agonists are in environmental, biological and synthetic materials remains to be determined. Using AhR-based assays, we demonstrate the presence of potent AhR agonists in a variety of common commercial and consumer items. Solvent extracts of paper, rubber and plastic products contain chemicals that can bind to and stimulate AhR DNA binding and/or AhR-dependent gene expression in hepatic cytosol, cultured cell lines, human epidermis and zebrafish embryos. In contrast to TCDD and other persistent dioxin-like HAHs, activation of AhR-dependent gene expression by these extracts was transient, suggesting that the agonists are metabolically labile. Solvent extracts of rubber products produce AhR-dependent developmental toxicity in zebrafish in vivo, and inhibition of expression of the metabolic enzyme CYP1A, significantly increased their toxic potency. Although the identity of the responsible AhR-active chemicals and their toxicological impact remain to be determined, our data demonstrate that AhR active chemicals are widely distributed in everyday products.     

Use of recombinant human ferrochelatase as a sensitive bioassay for N-alkylprotoporphyrin IX formed after interaction of porphyrinogenic xenobiotics with rat liver microsomes

Several porphyrinogenic xenobiotics elicit mechanism-based inactivation of cytochrome P450 (CYP) isozymes, leading to the formation of N-alkylprotoporphyrin IX (N-alkylPP), a potent inhibitor of ferrochelatase, the terminal enzyme in heme biosynthesis. Recognizing their role in experimental porphyria, our long term objective is the establishment of an appropriate in vitro system for the detection and quantification of N-alkylPPs, formed in human liver after the administration of potential porphyrinogenic compounds. In a previous study, we used a combination of thin-layer chromatography and UV-visible spectrophotometry to isolate and identify N-alkylPPs after incubating porphyrinogenic compounds with rat liver microsomes. However, the overall yield of N-alkylPPs was low, and it was concluded that in vitro systems, such as human lymphoblastoid microsomal preparations containing single cDNA-expressed human cytochrome P450 (CYP) isozymes, do not contain sufficient CYP for in vitro studies designed to isolate N-alkylPP. In the present study we demonstrate that purified recombinant human ferrochelatase (FC) provides an extremely sensitive bioassay system for N-alkylPPs and is capable of detecting N-alkylPP in the 10(-6) nmol range. Therefore, we propose that this bioassay system might allow the use of human lymphoblastoid microsomal preparations containing single cDNA-expressed human CYP isozymes to detect N-alkylPP produced after mechanism-based (catalysis-based) CYP inactivation. If this is found to be correct it will facilitate identification of potentially porphyrinogenic drugs prior to administration to humans.     

Lactobacillus bulgaricus OLL1181 activates the aryl hydrocarbon receptor pathway and inhibits colitis

Increasing evidence suggests that the aryl hydrocarbon receptor (AhR) pathway has an important role in the regulation of inflammatory responses. Most recently, we have shown that the activation of the AhR pathway by a potent AhR agonist inhibits the development of dextran sodium sulfate (DSS)-induced colitis, a model of human ulcerative colitis, by the induction of prostaglandin E2 (PGE2) in the large intestine. Because several strains of probiotic lactic acid bacteria have been reported to inhibit DSS-induced colitis by unidentified mechanisms, we hypothesized that particular strains of lactic acid bacterium might have the potential to activate the AhR pathway, thereby inhibiting DSS-induced colitis. This study investigated whether there are specific lactic acid bacterial strains that can activate the AhR pathway, and if so, whether this AhR-activating potential is associated with suppression of DSS-induced colitis. By using AhR signaling reporter cells, we found that Lactobacillus bulgaricus OLL1181 had the potential to activate the AhR pathway. OLL1181 also induced the mRNA expression of cytochrome P450 family 1A1 (CYP1A1), a target gene of the AhR pathway, in human colon cells, which was inhibited by the addition of an AhR antagonist, α-naphthoflavon (αNF). In addition, mice treated orally with OLL1181 showed an increase in CYP1A1 mRNA expression in the large intestine and amelioration of DSS-induced colitis. Thus, OLL1181 can induce activation of the intestinal AhR pathway and inhibit DSS-induced colitis in mice. This strain of lactic acid bacterium has therefore the potential to activate the AhR pathway, which may be able to suppress colitis.     

Lentinan from shiitake mushroom (Lentinus edodes) suppresses expression of cytochrome P450 1A subfamily in the mouse liver

Induction of xenobiotics metabolizing enzymes is related to the formation and chemoprevention of cancer. Since cytochrome P450s (CYPs) including CYP1A subfamily metabolize certain pro-carcinogens to their ultimate forms, down-regulation of CYP1As by food factors leads to the prevention of cancer. Mushroom polysaccharides, especially beta-glucans such as lentinan from Lentinus edodes, possess the anti-tumor and immunomodulating activities through the cytokine production from immunocytes. Recent our studies have demonstrated that lentinan suppresses hepatic CYP1As expression in the both constitutive and inducible levels through the production of tumor necrosis factor-alpha and an increase in the DNA-binding activity of nuclear factor-kappaB. This paper discusses on the effective lentinan dosage and route of administration for suppression of CYP1As.     

Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum)

Capsidiol is a bicyclic, dihydroxylated sesquiterpene produced by several solanaceous species in response to a variety of environmental stimuli. It is the primary antimicrobial compound produced by Nicotiana tabacum in response to fungal elicitation, and it is formed via the isoprenoid pathway from 5-epi-aristolochene. Much of the biosynthetic pathway for the formation of this compound has been elucidated, except for the enzyme(s) responsible for the conversion of 5-epi-aristolochene to its dihydroxylated form, capsidiol. Biochemical evidence from previous studies with N. tabacum (Whitehead, I. M., Threlfall, D. R., and Ewing, D. F., 1989, Phytochemistry 28, 775-779) and Capsicum annuum Hoshino, T., Yamaura, T., Imaishi, H., Chida, M., Yoshizawa, Y., Higashi, K., Ohkawa, H., Mizutani, J., 1995, Phytochemistry 38, 609-613. suggested that the oxidation of 5-epi-aristolochene to capsidiol was mediated by at least one elicitor-inducible cytochrome P450 hydroxylase. In extending these observations, we developed an in vivo assay for 5-epi-aristolochene hydroxylase activity and used it to demonstrate a dose-dependent inhibition of activity by ancymidol and ketoconazole, two well characterized inhibitors of cytochrome P450 enzymes. Using degenerate oligonucleotide primers designed to the well conserved domains found within most P450 enzymes, including the heme binding domain, cDNA fragments representing four distinct P450 families (CYP71, CYP73, CYP82, and CYP92) were amplified from a cDNA library prepared against mRNA from elicitor-treated cells using PCR. The PCR fragments were subsequently used to isolate full-length cDNAs (CYP71D20 and D21, CYP73A27 and A28, CYP82E1 and CYP92A5), and these in turn were used to demonstrate that the corresponding mRNAs were all induced in elicitor-treated cells, albeit with different induction patterns. Representative, full-length cDNAs for each of the P450s were engineered into a yeast expression system, and the recombinant yeast assessed for functional expression of P450 protein by measuring the CO difference spectra of the yeast microsomes. Only microsomal preparations from yeast expressing the CYP71D20 and CYP92A5 cDNAs exhibited significant CO difference absorbance spectra at 450 nm and were thus tested for their ability to hydroxylate 5-epi-aristolochene and 1-deoxycapsidiol, a putative mono-hydroxylated intermediate in capsidiol biosynthesis. Interestingly, the CYP71D20-encoded enzyme activity was capable of converting both 5-epi-aristolochene and 1-deoxycapsidiol to capsidiol in vitro, consistent with the notion that this P450 enzyme catalyzes both hydroxylations of its hydrocarbon substrate.     

Hepatic gene expression changes in mouse models with liver-specific deletion or global suppression of the NADPH-cytochrome P450 reductase gene. Mechanistic implications for the regulation of microsomal cytochrome P450 and the fatty liver phenotype

NADPH-cytochrome P450 reductase (CPR) is an essential component for the function of many enzymes, including microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. In liver-Cpr-null (with liver-specific Cpr deletion) and Cpr-low (with reduced CPR expression in all organs examined) mouse models, a reduced serum cholesterol level and an induction of hepatic P450s were observed, whereas hepatomegaly and fatty liver were only observed in the liver-Cpr-null model. Our goal was to identify hepatic gene expression changes related to these phenotypes. Cpr-lox mice (with a floxed Cpr gene and normal CPR expression) were used as the control. Through microarray analysis, we identified many genes that were differentially expressed among the three groups of mice. We also recognized the 12 gene ontology terms that contained the most significantly changed gene expression in at least one of the two mouse models. We further uncovered potential mechanisms, such as an increased activation of constitutive androstane receptor and a decreased activation of peroxisomal proliferator-activated receptor-alpha by precursors of cholesterol biosynthesis, that underlie common changes (e.g. induction of multiple P450s and suppression of genes for fatty acid metabolism) in response to CPR loss in the two mouse models. Additionally, we observed model-specific gene expression changes, such as the induction of a fatty-acid translocase (Cd36 antigen) and the suppression of carnitine O-palmitoyltransferase 1 (Cpt1a) and acyl-CoA synthetase long chain family member 1 (Acsl1), that are potentially responsible for the severe hepatic lipidosis and an altered fatty acid profile observed in liver-Cpr-null mice.     

Modulation of Ah receptor and CYP1A1 expression by alpha-naphthoflavone in rainbow trout hepatocytes

The objective of this study was to evaluate whether alpha-naphthoflavone (ANF) modulates aryl hydrocarbon receptor (AhR) signaling in rainbow trout (Oncorhynchus mykiss). AhR and cytochrome P450 1A1 (CYP1A1) protein and mRNA content were used as indictors of AhR signaling. Primary culture of rainbow trout hepatocytes were exposed to different concentrations of ANF (10(-9)-10(-5) M), while beta-naphthoflavone (BNF 10(-10)-10(-6) M) and a combination of ANF and BNF were used to elucidate the impact of ANF on AhR signaling. ANF increased AhR and CYP1A1 protein expression in a concentration-related manner; the maximal induction was about 50% that of BNF. Despite the differences in protein content between ANF and BNF stimulation, the maximal AhR and CYP1A1 mRNA abundance seen with the high concentrations of ANF and BNF were similar. ANF significantly decreased ( approximately 50%) BNF-induced AhR protein expression (only at 10(-9) M), but not CYP1A1 protein and gene expression. In addition, ANF at a sub-maximal concentration (10(-7) M) did not affect BNF-induced AhR protein content, but increased the sensitivity of hepatocytes to BNF-mediated CYP1A1 protein expression. Taken together, the mode of action of ANF appears similar to BNF, including modulation of AhR expression and activation of AhR-mediated signaling in rainbow trout hepatocytes. Overall, ANF is not only a partial AhR agonist, but may also modify BNF-mediated AhR signaling in trout hepatocytes.